A method for detecting thrombin based on nafion-modified glassy carbon electrode
A glassy carbon electrode and thrombin technology, which is applied in biochemical equipment and methods, microbial determination/inspection, measurement devices, etc., can solve the problems of complicated thrombin detection methods, large consumption of monomer samples, etc. Hardware pre-preparation process, avoiding the DNA modification process, and the effect of strong practicability
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Embodiment 1
[0034] The making of the glassy carbon electrode modified by embodiment 1 Nafion
[0035] The glassy carbon bare electrode was ground and polished with 0.3 nm and 0.05 nm aluminum powder in turn, and the CV curve was scanned in the potassium ferricyanide solution. The peak voltage difference was 80-100 mV, and it was dried for later use. The concentration of Nafion used was 0.01wt.%, and the drop volume was 3.5 µL. Add the Nafion solution to the center of the glassy carbon electrode dropwise, let it diffuse naturally, place it at room temperature, and volatilize naturally to form a uniform Nafion film on the glassy carbon electrode, and then a negatively charged Nafion-modified glassy carbon working electrode can be obtained. figure 1 Schematic diagram of Nafion modified glassy carbon electrode; figure 2 Impedance diagrams before and after Nafion modification.
Embodiment 2
[0036] Example 2 Quantitative detection of thrombin
[0037]The total volume of the reaction system was 100 µL. 5 mM Tris-HCl buffer containing 1 mM MgCl 2 , 10 mM NaCl, 1 mM DTT (dithiothreitol), and pH 7.5) as a solvent mixed with 1 µM thrombin aptamer DNA strand (5′-GTGGTAGGGCAGGTT GGGGTGACT-3′) and methylene blue-labeled Complementary DNA strands (5′-AGTCACCCCAACCTGCCCTACCAC-Methylene blue (MB)-3′) were hybridized at a constant temperature of 37°C for 1 hour to form a double-stranded DNA structure. Because the phosphate backbone of DNA is negatively charged, and its negative charge is related to the number of bases contained. The more bases there are, the stronger the negative charge. When there is no target thrombin in the solution, add 20 U of Exo I enzyme, because the solution only contains double-stranded DNA structure (the structure contains DNA strands modified with methylene blue), Exo I enzyme cannot cut it, the double-stranded The negative charge of the DNA st...
Embodiment 3
[0039] Embodiment 3 thrombin detection linearity
[0040] The total volume of the reaction system was 100 µL. 5 mM Tris-HCl buffer containing 1 mM MgCl 2 , 10 mM NaCl, 1 mM DTT (dithiothreitol), and pH 7.5) as a solvent, mix 1 µM thrombin aptamer DNA strand and complementary DNA strand labeled with methylene blue, and react at a constant temperature of 37°C Hybridization was performed for 1 hour to form a double-stranded DNA structure. Then add different concentrations of thrombin to react for 1 h to break away from the thrombin aptamer DNA chain, and then add 20 U of Exo I enzyme to specifically cut the complementary DNA single strand marked with methylene blue, and then release the short-chain DNA DNA fragments with methylene blue. The Nafion-modified glassy carbon electrode was inserted into the reaction solution, and the DPV differential pulse electrical signal was collected using an electrochemical workstation.
[0041] After detection, in the range of 0.01 nM ~ 1000 ...
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